Separating components of biological fluids and tissues is often necessary for clinical diagnostic procedures, scientific research, and occasionally treatment of patients. In the clinical diagnostics field, for example, there is a need for compositions and methods which permit rapid isolation of purified lymphocytes for tissue typing procedures, immunologic function tests, and various other procedures. Basic research also requires purified lymphocytes as well as other cell types from blood. In addition, studies on cultured cells and subcellular components such as plasmids, DNA, chromosomes, mitochondria and other subcellular components also require highly purified preparations.
Separation and purification might be effected in several ways. However, since the isolated cells are often used in procedures which require viable cells, it is important that the functions of the cells so isolated be unimpaired. To insure viability of the cells and unimpaired biological function of cells and subcellular components, it is important to avoid introducing possible interfering substances in the course of the separation procedure. For example, isolated lymphocytes used in histocompatibility tests are stimulated with various mitogens. The medium used must not in itself be a mitogen since this will affect the validity of the measurements of DNA synthesis.
Density gradient centrifugation is one technique used for separation of biological cells and subcellular components. It is highly desirable that the material chosen for formation of the gradient have certain characteristics which will impart compatability with sensitive biological materials. Gradient materials which have been employed in the art include sucrose, dextran, bovine serum albumin (BSA), Ficoll (registered trademark of Pharmacia), iodinated low molecular weight compounds such as Metrizamide and heavy salts such as cesium chloride. Most of these materials, however, have undesirable characteristics which potentially may impair the biochemical functions of the desired isolated fractions. For example, some currently available blood separation media contain erythrocyte aggregation polmers which may decrease mitogen responsiveness of isolated lymphocyte preparations (J. Immunol. Meth. 38:43-51, 1980). These materials may also form solutions of undesirably high osmolality or viscosity. Cell aggregation is often caused by BSA at physiological pH (7.4) and it is undesirable to employ reduced pH (5.1) because it introduces other problems such as cell swelling (which alters cell density) and possible impairment of cell function. It is also expensive for use in large scale separations. Ficoll may similarly cause cellular aggregation which can be remedied only by use of a dispersing agent, or undesirably, lowering of the pH. Ficoll is also highly viscous, making it difficult to generate a linear isoosmotic gradient with it. It is also difficult to separate cells with very similar buoyant densities with any of these materials, even with the use of discontinuous gradients.
A density gradient material which has been used with some success for cell separation is colloidal silica. Colloidal silica is an aqueous suspension of colloidal particles formed by polymerization of monosilicic acid from SiO.sub.2 dissolved in water. Individual particles average 130-140 .ANG. in size and generally range from about 30 to 220 .ANG.. The colloidal suspension is most stable for storage at pH 8-10 at which the colloidal particles have a net negative charge. It is, however, not entirely satisfactory. Colloidal silica solutions are irreversibly precipitated on freezing and form gels in the presence of proteins under certain ionic conditions such as above 0.1M NaCl at pH 5-7. Unmodified silica gels also exhibit toxicity towards a number of cell types including macrophages and red blood cells. Numerous attempts have been made to reduce this toxicity and to increase the stability of the colloid in salt solutions and protein at physiological pH by coating the colloidal silica with a polymer. Polymers such as dextran (Exp. Cell Res., 50: 355-368 (1968)), polyvinyl alcohol (PVA) (J. Coll. and Interface Sci., 51: 388-393 (1974)), polyethylene glycol (PEG) ( Exp. Cell Res., 57: 338-350 (1969), Arch. Biochem. Biophys., 168: 289-301 (1975)), dextran sulfate, methyl cellulose, carboxymethyl cellulose (Exp. Cell Res., 50: 355-368, (1968)), polyvinylpyrrolidone (PVP) (Exp. Cell Res., 46: 621-623 (1966); Exp. Cell Res., 50: 355-368 (1968); J. Cell Biol., 55: 579-585 (1972); Exp. Cell Res., 110: 449-457 (1977)) and a mixture of PEG, BSA and Ficoll (Arch. Biochem. Biophys., 168: 289-301 (1975)) have been used as coatings for silica sols such as Ludox (registered trademark of DuPont).
Merely coating the silica particles with polymer also presents problems. The coating procedure requires the use of an excess of free polymer in the solution. The excess polymer increases the osmolality and viscosity of the solution. It is also difficult to remove the polymer from the purified biological material. Furthermore, while the morphological characteristics of cells and organelles purified with polymer-coated colloidal silica are generally acceptable, the biochemical characteristics are often imparied. For example, lymphocytes isolated with mixtures of colloidal silica and PVP have a decreased incorporation of radioactive thymidine as compared to cells in control medium. Exp. Cell Res., 50: 353 (1968).
Another approach to reducing the toxicity of colloidal silica has been to chemically modify the surface of the silica particles. An example of a chemically modified colloidal silica is Ludox AM (registered trademark of DuPont) in which aluminum is chemically incorporated into the colloidal silica. J. Colloid and Interface Science, 55:25 (1975). This preparation is reportedly stable over a wide pH range, however it is not suitable for cell separation unless it is first extensively dialysed and/or treated with charcoal to render it nontoxic to lymphoid cells and useful for separating lymphocyte subpopulations. J. Immunological Methods, 28:277 (1979). Ludox AM reportedly makes only a minor contribution to osmolality and it is therefore possible to construct isoosmotic gradients using this compound. Ludox AM has several drawbacks, however. The gradient material must be stored under sterile conditions since it will support the growth of various microorganisms. Antibiotics and antifungal agents must be added to the gradient material to inhibit the growth of contaminating microorganisms which may be found in various lymphoid tissues. Furthermore, in order to adequately separate subpopulations of lymphoid cells it is necessary to use discontinuous gradients. J. Immunological Methods, 28:277-292 (1979). Limitations inherent in discontinuous density separation (Ann. Rev. Biophys. Bioeng. 1:93-130, 1972; Int. Rev. Exp. Path 14:91-204, 1975; "Automated Cell Identification and Cell Sorting", Academic Press, pp. 21-96, 1970) may contribute to lower lymphocyte recoveries, which may result in altered lymphocyte ratios (Scand. J. Immunol. 3:61, 1974; Clin. Immunol. Immunopath. 3:584-597, 1975). Furthermore, discontinuous gradients are made from individual solutions of varying densities. It is extremely time-consuming to make up the various solutions of the correct density. Generation of density gradients by centrifugation also requires speeds of 20,000 to 30,000 rpm.
Another chemically modified colloidal silica which is commercially available is Percoll (registered trademark of Pharmacia) which is a silica particle to which a layer of PVP is hydrogen bonded Anal. Biochem., 88: 271-282 (1978). Percoll has been used widely for separating blood cell components as well as subcellular organelles from a variety of sources. As supplied by the manufacturer, Percoll has a density of 1.130.+-.0.005 g/cm.sup.3, a pH of 9.0.+-.0.5 and an osmolality of &lt;25 mOsM/kg. The Percoll is made isoosmotic by adding physiological saline and adjusting the pH to 7.0-7.4 by adding acid or base. The density must also be carefully adjusted. If cells have a buoyant density greater than 1.11-1.12 g/cm.sup.3 then the Percoll must be concentrated. The usual technique for using Percoll is either to preform the gradient by layering or by using a gradient former or by centrifugation (generally requires 20,000 to 30,000 rpm) prior to addition of the sample. Alternatively, the cell preparation may be mixed directly with diluted isoosmotic Percoll prior to centrifugation.
Several factors limit the ease, performance and utility of Percoll as a blood cell separation medium, however. Percoll has a strong absorbency in the ultraviolet region due to the PVP. This is a significant disadvantage when density gradients are analyzed for nucleic acids and proteins by spectrophotometric methods. PVP also gives a high background in the Lowry method for protein determination. Cell Separation: Methods and Selected Applications, Vol I. 115, 134 (1982). Percoll is somewhat stable at physiological pH and ionic strength, however it is not stable to autoclaving after it is made isoosmotic (by the addition of salt, acid and base), and in dilute solutions it tends to aggregate. The latter problem is due to dissociation of PVP from the silica surface and can be prevented by the addition of low concentrations of free PVP. As noted above, free PVP has a negative effect on at least some cell functions. It is also difficult to separate cells which have only small differences in buoyant density with Percoll.
It has now been found that a novel colloidal silica preparation useful for the separation of biological materials can be prepared by using a novel chemical modification technique. The composition is made by reacting an organosilane under aqueous conditions with non-porous colloidal silica at an elevated temperature and at alkaline pH to form a covalent linkage between the silica particle and the organosilane. The composition may be used for isolating specific cell types or specific subcellular components based upon their buoyant density. The composition provides for improved purity of recovered cells and subcellular components and for decreased sample processing time. A composition which combines different sizes of reagent-modified silica particles can also be prepared. This combination composition offers the further advantage of separating cells and cellular components with only minute differences in buoyant density. This type of separation has up until now been impossible or impractical to achieve. A further composition which can be prepared consists of reagent-modified silica particles to which purified antibody has been coupled. This antibody-modified composition offers the advantage of separating cells and cell components based upon their antigenic determinants as well as their complement of any other cellular component, such as peptide hormone receptors, to which antibodies can be raised.
The composition has several desirable characteristics. The reagent modification reduces the toxicity of the colloidal silica and eliminates aggregation of the colloidal silica particles in the presence of physiological salt and protein. The composition is compatible with biological material and is suitable for density separation of both cellular and subcellular biological particles. It permits the use of either preformed gradients or in situ density gradient formation and rapid cell separation using relatively low-speed centrifugation. The composition is of physiological ionic strength and pH, isoosmolar, of low viscosity, and in a density range of 1.0 to 1.4 g/cm.sup.3. It is stable over a wide range of temperature and pH values. As evidence of its stability, the composition is completely stable to autoclaving at physiological conditions. Furthermore, it is soluble or dispersible in aqueous solutions, is easily removed from biological specimens, and is of low cost.
The methods for use of this composition also offer several advantages. Traditional blood separation procedures require careful blood layering technique. The technical skill required to perform blood separation with this composition is much less than with other techniques. For example, no layering is required with the in situ technique. The material to be separated is simply mixed with the reagent-modified colloidal silica and centrifuged. To separate mononuclear cells from whole blood, the inherent density difference which exists between mononuclear cells and other cellular elements present in peripheral blood is used. A continuous density gradient suitable for mononuclear cell separation is formed upon centrifugation by the sedimentation of the reagent-modified colloidal silica particles. Rapid gradient formation is due to the high sedimentation rate of the particles used (.gtoreq.200 .ANG. diameter) for this type of separation. Cell separation is accomplished by the movement of the peripheral blood cells during centrifugation to their respective buoyant densities within the continuous density gradient. The separation technique is inexpensive and requires little if any specialized equipment other than a clinical centrifuge.